Visualizing Dynamic Actin Crosslinking Processes Driven by the Actin Binding Protein Anillin

Abstract

Anillin is an actin binding protein that can bundle actin filaments and associate with the plasma membrane in the ring-like actin network. Anillin can be divided into a membrane-bound domain and an actin binding domain (ABD). Although the ABD in anillin plays an important role in the dynamic ring transition, it is unclear how the ABD interacts with the actin ring structures during the contractile ring to midbody ring transition. In order to elucidate the underlying intermolecular interactions between the ABD and actin filaments, it is necessary to observe dynamic crosslinking processes. Here, we performed real-time imaging of actin filament crosslinking dynamics by the ABD of anillin using total internal reflection fluorescence microscopy (TIRFM) and high-speed atomic force microscopy (Hs-AFM) for single-molecule imaging. Both human full-length anillin and anillin N-terminal ABD constructs existed as monomers and showed relatively low binding affinity for actin filaments. Hs-AFM showed that anillin monomers crosslinked with actin filaments at a distance of 8 nm. The distance between the aligned crosslinked anillin molecules was approximately 37 nm, which corresponded to the half pitch of an actin filament. Furthermore, the polarity of these filaments was both parallel and antiparallel. Our direct and dynamic high-resolution visualization of the actin crosslinking process provides a powerful new approach to studying the interactions and dynamics of biomolecules.